International Journal of Biomedical Research

POLYMERASE CHAIN REACTION: METHODS, PRINCIPLES AND

APPLICATION
Dr.Mohini Joshi1*, Dr.Deshpande J.D2.
1

Department of Anatomy, Rural Medical College, Pravara Institute of Medical Sciences,

Loni, Maharashtra, India
2
Department of Community Medicine,Rural Medical College, Pravara Institute of Medical
Sciences, Loni, Maharashtra, India
Corresponding author*: atharvamohini@gmail.com
This article is available online at www.ssjournals.com
ABSTRACT
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to
amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence. PCR is now a
common and often indispensable technique used in medical and biological research labs for
a variety of applications. There are three major steps involved in the PCR technique:
denaturation, annealing, and extension. PCR is useful in the investigation and diagnosis of a
growing number of diseases. Qualitative PCR can be used to detect not only human genes
but also genes of bacteria and viruses. PCR is also used in forensics laboratories and is
especially useful because only a tiny amount of original DNA is required. PCR can identify
genes that have been implicated in the development of cancer. Molecular cloning has
benefited from the emergence of PCR as a technique. The present paper is an attempt to
review basics of PCR.
KEY WORDS: PCR, Principles, Application
INTRODUCTION
The polymerase chain reaction (PCR) is a
scientific technique in molecular biology
to amplify a single or a few copies of a
piece of DNA across several orders of
magnitude, generating thousands to
millions of copies of a particular DNA
sequence. Polymerase Chain Reaction
was developed in 1984 by the American
biochemist, Kary Mullis. Mullis received
the Nobel Prize and the Japan Prize for
developing PCR in 1993.1 However the
basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971.
Progress was limited by primer synthesis

and polymerase purification issues.2 PCR

is now a common and often indispensable
technique used in medical and biological
research labs for a variety of
applications.3 The polymerase chain
reaction is a powerful technique that has
rapidly become one of the most widely
used techniques in molecular biology
because it is quick, inexpensive and
simple. The technique amplifies specific
DNA fragments from minute quantities of
source DNA material, even when that
source DNA is of relatively poor quality.4
PCR; the quick, easy method for
generating unlimited copies of any

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Joshi et al
fragment of DNA, is one of those
scientific developments that actually
deserve timeworn superlatives like
"revolutionary" and "breakthrough. From
the daily practicalities of medical
diagnosis to the theoretical framework of
systematics, from courts of law to field
studies of animal behavior, PCR takes
analysis of tiny amounts of genetic
material-even damaged genetic materialto a new level of precision and reliability.
Furthermore,
many
important
contributions to the development and
application of PCR technology have been
made; however the present paper is an
attempt to review basics of PCR.
Basic concept of PCR
The basic PCR principle is simple. As the
name implies, it is a chain reaction: One
DNA molecule is used to produce two
copies, then four, then eight and so forth.
This continuous doubling is accomplished
by
specific
proteins
known
as
polymerases, enzymes that are able to
string together individual DNA building
blocks to form long molecular strands. To
do their job polymerases require a supply
of DNA building blocks, i.e. the
nucleotides consisting of the four bases
adenine (A), thymine (T), cytosine (C)
and guanine (G). They also need a small
fragment of DNA, known as the primer,
to which they attach the building blocks
as well as a longer DNA molecule to
serve as a template for constructing the
new strand. If these three ingredients are
supplied, the enzymes will construct exact
copies of the templates. PCR is a method
used to acquire many copies of any
particular strand of nucleic acids. Its a
means of selectively amplifying a
particular segment of DNA. The segment
may represent a small part of a large and
complex mixture of DNAs e.g. a specific
exon of a human gene. It can be thought

Review Article

of as a molecular photocopier. PCR can

amplify a usable amount of DNA (visible
by gel electrophoresis) in ~2 hours. The
template DNA need not be highly purified
a boiled bacterial colony. The PCR
product can be digested with restriction
enzymes, sequenced or cloned. PCR can
amplify a single DNA molecule, e.g. from
a single sperm. The polymerase chain
reaction relies on the ability of DNAcopying enzymes to remain stable at high
temperatures. PCR has transformed the
way that almost all studies requiring the
manipulation of DNA fragments may be
performed as a result of its simplicity and
usefulness.5 In Mullis's original PCR
process, the enzyme was used in vitro.
The double-stranded DNA was separated
into two single strands of DNA by heating
it to 96C. At this temperature, however,
the E.Coli DNA polymerase was
destroyed, so that the enzyme had to be
replenished with new fresh enzyme after
the heating stage of each cycle. Mullis's
original PCR process was very inefficient
since it required a great deal of time, vast
amounts of DNA-Polymerase, and
continual attention throughout the PCR
process.6
Steps in PCR
There are three major steps involved in
the
PCR
technique:
denaturation,
annealing, and extension. In step one; the
DNA is denatured at high temperatures
(from 90 - 97 degrees Celsius). In step
two, primers anneal to the DNA template
strands to prime extension. In step three,
extension occurs at the end of the
annealed
primers
to
create
a
complementary copy strand of DNA. This
effectively doubles the DNA quantity
through the third steps in the PCR cycle.
To amplify a segment of DNA using PCR,
the sample is first heated so the DNA
denatures, or separates into two pieces of

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Joshi et al
single-stranded DNA. Next, an enzyme
called "Taq polymerase" synthesizes builds - two new strands of DNA, using
the original strands as templates. This
process results in the duplication of the
original DNA, with each of the new
molecules containing one old and one new
strand of DNA. Then each of these strands
can be used to create two new copies, and
so on, and so on.7 The annealing phase
happens at a lower temperature, 50-60C.
This allows the primers to hybridize to
their respective complementary template
strands, a very useful tool to forensic
chemistry. The newly-formed DNA strand
of primer attached to template is then used
to create identical copies off the original
template strands desired. Taq polymerase
adds available nucleotides to the end of
the annealed primers. The extension of the
primers by Taq polymerase occurs at
approx 72C for 2-5 minutes. DNA
polymerase I cannot be used to elongate
the primers as one would expect because
it is not stable at the high temperatures
required for PCR. The beauty of the PCR
cycle and process is that it is very fast
compared to other techniques and each
cycle doubles the number of copies of the
desired DNA strand. After 25-30 cycles,
whoever is performing the PCR process
on a sample of DNA will have plenty of
copies of the original DNA sample to
Y

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conduct experimentation. Assuming the

maximum amount of time for each step,
30 cycles would only take 6 hours to
complete.
As the process of denaturation, annealing,
and polymerase extension is continued the
primers repeatedly bind to both the
original
DNA
template
and
complementary sites in the newly
synthesized strands and are extended to
produce new copies of DNA. The end
result is an exponential increase in the
total number of DNA fragments that
include the sequences between the PCR
primers, which are finally represented at a
theoretical abundance of 2n, where n, is
the number of cycles.5, 8
Due to the introduction of a thermostable
DNA polymerase, the Taq DNA
polymerase once, at the beginning of the
PCR
reaction.9 The
thermostable
properties of the DNA polymerase
activity were isolated from Thermus
aquaticus (Taq) that grow in geysers of
over 110C, and have contributed greatly
to the yield, specificity, automation, and
utility of the polymerase chain reaction.
The Taq enzyme can withstand repeated
heating to 94C and so each time the
mixture is cooled to allow the
oligonucleotide primers to bind the
catalyst for the extension is already
present.10 After the last cycle, samples are
usually incubated at 72C for 5 minutes to
fill in the protruding ends of newly
synthesized PCR products. To ensure
success, care should be taken both in
preparing the reaction mixture and setting
up the cycling conditions. Increasing the
cycle number above ~35 has little positive
effect because the plateau occurs when the
reagents are depleted; accumulate. The
specificity of amplification depends on
the extent to which the primers can
recognize and bind to sequences other
than the intended target DNA sequences.

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Joshi et al
METHODS
In
molecular
biology,
real-time
polymerase chain reaction, also called
quantitative real time polymerase chain
reaction is a laboratory technique based
on the PCR, which is used to amplify and
simultaneously quantify a targeted DNA
molecule.
Traditionally,
PCR
is
performed in a tube and when the reaction
is complete the products of the reaction
(the amplified DNA fragments) are
analyzed and visualized by gel
electrophoresis. However, Real-Time
PCR permits the analysis of the products
while the reaction is actually in progress.
This is achieved by using various
fluorescent dyes which react with the
amplified product and can be measured by
an instrument. This also facilitates the
quantitation of the DNA. Quantitative
PCR (Q-PCR), as this technique is known,
is used to measure the quantity of a PCR
product (usually in a real-time PCR
procedure). It is the method of choice to
quantitatively measure starting amounts of
DNA, cDNA or RNA. PCR is therefore
often used to determine whether a DNA
sequence is present in a sample and the
number of its copies in the sample.
Another advantage of Real-Time PCR is
rapidity of the assay, since it is not
necessary to perform electrophoresis or
other procedure after the DNA
amplification reaction. 11, 12 Digital PCR
concept was conceived in 1992 by Sykes
et al.13 It is a refinement of conventional
polymerase chain reaction methods that
can be used to directly quantify and
clonally amplify nucleic acids including
DNA, cDNA or RNA. The key difference
between dPCR and traditional PCR lies in
the method of measuring nucleic acids
amounts, with the former being a more
precise method than PCR. PCR carries out
one reaction per single sample. dPCR also
carries out a single reaction within a

Review Article

sample, however the sample is separated

into a large number of partitions and the
reaction is carried out in each partition
individually. This separation allows a
more reliable collection and sensitive
measurement of nucleic acid amounts.14
Inverse PCR is a variant of the
polymerase chain reaction that is used to
amplify DNA with only one known
sequence. One limitation of conventional
PCR is that it requires primers
complementary to both termini of the
target DNA, but this method allows PCR
to be carried out even if only one
sequence is available from which primers
may be designed.15 Nested polymerase
chain reaction is a modification of
polymerase chain reaction intended to
reduce the contamination in products due
to the amplification of unexpected primer
binding sites.16 Touchdown polymerase
chain reaction is a method of polymerase
chain reaction by which primers will
avoid amplifying nonspecific sequences.
The earliest steps of a touchdown
polymerase chain reaction cycle have high
annealing temperatures. The annealing
temperature is decreased in increments for
every subsequent set of cycles.17
Applications of PCR
PCR is helping in the investigation and
diagnosis of a growing number of
diseases. It has also long been a standard
method in all laboratories that carry out
research on or with nucleic acids. Even
competing techniques such as DNA chips
often require amplification of DNA by
means of PCR as an essential preliminary
step. The polymerase chain reaction is
used by a wide spectrum of scientists in
an ever-increasing range of scientific
disciplines. The use of reverse
transcriptases to evaluate RNA levels and
the extension of PCR technology to
quantify DNA amplification in real time

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Joshi et al
has brought major advances to the
application of PCR. By allowing the
determination and quantification of
changes in gene expression, these
techniques have provided a greater
understanding of disease processes and
now serve as a foundation for diagnostics
and basic science research.18 In
microbiology and molecular biology, for
example, PCR is used in research
laboratories in DNA cloning procedures,
Southern blotting, DNA sequencing,
recombinant DNA technology, to name
but a few. In clinical microbiology
laboratories PCR is invaluable for the
diagnosis of microbial infections and
epidemiological studies. PCR is also used
in forensics laboratories and is especially
useful because only a tiny amount of
original DNA is required, for example,
sufficient DNA can be obtained from a
droplet of blood or a single hair. In fact, a
number of trials using PCR for detection
of a broad range of bacteria in CSF
specimens have been reported.19-21 Since
the culture of C. pneumoniae is difficult in
most clinical laboratories, determination
of this bacterium in clinical specimens has
been widely performed using the PCR
technique even though there is no
standardized PCR method for detection of
this organism. Nested PCR is one of these
protocols for detection of only a few
bacteria in clinical specimens.22
Qualitative PCR can of course be used to
detect not only human genes but also
genes of bacteria and viruses. One of the
most important medical applications of
the classical PCR method is therefore the
detection of pathogens. Many viruses
contain RNA rather than DNA. In such
cases the viral genome has to be
transcribed before PCR is performed, and
RTPCR is therefore used. Sometimes it is
also necessary to detect pathogens outside
the body. Fortunately, the PCR method
can detect the DNA of microorganisms in

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any sample, whether of body fluids,

foodstuffs or drinking water. Quantitative
PCR provides additional information
beyond mere detection of DNA. It
indicates not just whether a specific DNA
segment is present in a sample, but also
how much of it is there. This information
is required in a number of applications
ranging from medical diagnostic testing
through target searches to basic research.
Another
important
application
of
quantitative PCR is in molecular
diagnosis, i.e. the diagnosis of diseases
based on molecular findings rather than
on physiological symptoms. In this
connection the diagnosis of viral diseases
is an area that is gaining increasing
importance. PCR is the most sensitive test
for herpes simplex virus, varicella-zoster
virus,
and
human
papillomavirus
infections.
Other
diagnostic
uses,
including tests for genetic diseases,
cancers, and other infectious diseases, are
evolving.23Another important application
in which quantitative PCR is used in the
field of infectious diseases is AIDS. It can
detect the AIDS virus sooner during the
first few weeks after infection than the
standard ELISA test.
Genetic factors are always involved in the
development of cancer. Their contribution
varies greatly depending on the type of
cancer. Genes not only help to determine
progression of the disease but can also
have a substantial influence on the
effectiveness of the available treatments.
Identifying the genes that play a role in
the development of cancer is therefore an
important step towards improving
treatment.
Both
qualitative
and
quantitative PCR play a crucial role in the
fight against cancer. PCR can identify
genes that have been implicated in the
development of cancer. There are
numerous applications for real-time
polymerase chain reaction in the
laboratory. It is commonly used for both

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Joshi et al
basic research and is deployed as a tool to
detect newly emerging diseases, such as
flu, in diagnostic tests. Digital PCR has
many potential applications, including the
detection and quantification of low-level
pathogens, rare genetic sequences, copy
number variations, and relative gene
expression in single cells. Clonal
amplification enabled by single-step
digital PCR is a key factor in reducing the
time and cost of many of the "nextgeneration sequencing" methods and
hence enabling personal genomics.14
Inverse PCR is especially useful for the
determination of insert locations. For
example, various retroviruses and
transposons randomly integrate into
genomic DNA. To identify the sites where
they have entered, the known, "internal"
viral or transposon sequences can be used
to design primers that will amplify a small
portion of the flanking, "external"
genomic DNA.15 Nested polymerase
chain reaction is a key part of many
genetics research laboratories, along with
uses in DNA fingerprinting for forensics
and other human genetic cases.
Conventional PCR requires primers
complementary to the termini of the target
DNA. A commonly occurring problem is
primers binding to incorrect regions of
the DNA, giving unexpected products.16
RT-PCR is commonly used in studying
the genomes of viruses whose genomes
are composed of RNA, such as Influenza
virus A and retroviruses like HIV. PCR
can be used for the diagnosis of Indian
visceral
leishmaniasis
with
great
accuracy. PCR can also be employed with
significant precision to predict cure of the
disease.24 Molecular cloning has benefited
from the emergence of PCR as a
technique.
Direct cloning was first
conducted using a DNA fragment
amplified by PCR and oligonucleotide
primers which contained restriction

Review Article

endonuclease recognition sites added to

their 5 ends. 25
CONCLUSION
The advancement of science has
transformed our lives in ways that would
have been unpredictable just a halfcentury ago. Molecular methods have
shown a promise in this aspect. PCR and
its applications hold scientific and
medical promise. PCR has very quickly
become an essential tool for improving
human health and human life. PCR has
completely revolutionized the detection of
RNA and DNA viruses. PCR is valuable
as a confirmatory test. PCR is a rapid
technique with high sensitivity and
specificity. PCR has also been credited to
have been able to detect mixed infections
with ease in many studies. PCR, a more
sophisticated
technique,
requires
infrastructural support, is expensive but
nevertheless, one cannot discount its
utilitarian advantages which are many
compared to the existing conventional
diagnostic methods.
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